Bring reagents at Room Temperature before use
Sample
Fresh serum free of haemolysis; heparinized Plasma
Alkaline Phosphatase
Kinetic Colorimetric Optimized Method
Principle
Reaction principle
Kinetic determination of ALKALINE PHOSPHATASE
according DGKC recommendation:
ALP
p - Nitrophenylphosphate + H2O
p - Nitrophenol + phosphate
The Rate of Absorbance increase, determined
photometrically at 405 nm, is direct proportional to ALP
activity in the sample.
*Reagents composition, Contents and Safety warnings
1. Buffer /enzyme reagent
DEA* Buffer
pH 9.8
1 mol/l
Magnesium Chloride
1 mmol/l
Sodium Azide
< 0.1%
* Diethanolamine
2. Coenzyme Reagent
p - Nitrophenylphosphate
40 mmol/l
The Buffer is classified as Xn = Harmful
Content: DIETHANOLAMINE
Risk:
R41 Risk of serious damage to eyes
R48/22 Harmful: danger of serious damage to health by
prolonged exposure if swallowed
S26 In case of contact with eyes, rinse immediately with
plenty of water and seek medical advise
S36/37/39 Wear suitable protective clothing, gloves and
eye/face protection
Storage and Stability of Reagents
Store the kit at 2 - 8°C
All the components are stable until the stated expiration
date if stored tightly closed and refrigerated
*Preparation and Stability of Working solution
Reagents:
liquid and ready to use
1- REAGENT PROCEDURE
Mix 4 volumes of Buffer with 1 volume of Substrate
Working solution stability: 10 days at 2 - 8° C.
2 - REAGENTS PROCEDURE
Ready to use
Safety precaution
For in vitro diagnostic use only.
Do not pipette by mouth.
Exercise the normal precautions required for handling
laboratory reagents.
*Procedure
1. Wavelength:
Hg 405 nm
2. Cuvette
light path: 1 cm
3. Temperature
25/30/37°C.
4. Adjust the instrument to zero against distilled water
One Reagent Procedure
Sample
20 µl
Two Reagents Procedure
Buffer
800 µl
Sample
20 µl
Working Solution
1 ml
Substrate
200 µl
Mix, wait 1 minute, read the Absorbance and start the
stopwatch simultaneously.
Read again after 1, 2 and 3 minutes.
Calculate the average absorbance difference per minute
(A/min)
Calculation
ALP (U/I) = A/min x 2759
Reference values
25°C
30°C
37°C
Adults
60 - 170 U/l
72 - 205 U/l
94 - 266 U/l
Children
151 - 471 U/l
182 - 567 U/l
236 - 736 U/l
These values should only be used as a guideline.
Each laboratory should establish its Normal Reference
Range
Performance Characteristics
A. LINEARITY LIMIT
The reaction is linear up to 2000 U/I
For higher value dilute sample 1:2 with normal saline,
repeat the test and multiply the value by 2
B. DETECTION LIMIT
Values less than 5 U/I give non - reproducible results
C. INTRA - ASSAY PRECISION (N = 20 replicates)
Mean (U/l)
SD
CV%
Control 1
169
3.36
1.98
Control 2
450
13.87
3.08
D. INTER - ASSAY PRECISION (20 replicates for 3 days)
Mean (U/l)
SD
CV%
Control 1
Control 2
169.7
449.7
4.31
7.17
2.54
1.59
E. ACCURACY
Comparation between this method (y) and another
commercial one (x), gave the following results:
N = 20
r = 0.99709
y=1
x + 15.5
F. INTERFERENCES
1. Haemoglobin up to 200 mg/dl does not interfere
2. Bilirubin up to 40 mg/dl does not interfere
3. Triglycerides up to 1500 mg/dl do not interfere
4. Ascorbic acid up to 40 mg/dl does not interfere
Quality Control
Control sera are recommended to monitor the performance
of manual and automated assay procedures.
Bibliography
1. Deutsche Gesellschaft for Klinische Chemie (DGKC)
Recommendation of the German Society of Clinical
Chemistry Standardization of methods for measurement
of enzymatic activities in biological fluids.
2. Z. Klin, Chem. & Klin, Biochem, 10, 182 (1972)
3. Kubler W., Symp. D. Deutsch Ges fur Lab. Med 8 (1973)
4. Fischbach F., Zawta B.: Klin. Lab. 38 555 (1992)
5. Szasz G. et al: Z. Kindrheilk, 111, 233 (1971)
6. Kaplan L.A., Pesce A. J.: Clinical Chemistry Mosby Ed.
(1996)
For in vitro diagnostic use only.
The following symbols are used on labels
For in vitro diagnostic use
Use by (last day of the month)
Temperature limitation
Batch Code